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Development of a High-Throughput Method for Quantification of Plasmopara viticola DNA in Grapevine Leaves by Means of Quantitative Real-Time Polymerase Chain Reaction

First, second, third, and sixth authors: Plant Pathology Group, Institute of Plant Sciences, Swiss Federal Institute of Technology, 8092 Zürich, Switzerland; fourth author: Plant Protection Operative Unit, Istituto Agrario di S. Michele all'Adige, via E. Mach 1, 38010 S. Michele all'Adige (TN), Italy; and fifth author: SafeCrop Centre, Istituto Agrario di San Michele all'Adige, via E. Mach 1, 38010 S. Michele all'Adige (TN), Italy

Plasmopara viticola is a strictly biotrophic oomycete that causes downy mildew, which is one of the most important grapevine diseases. Control of the disease is most often achieved by fungicide applications, which may have severe environmental consequences. Therefore, alternative control strategies based on biocontrol agents (BCAs) are currently in development. Thousands of potential BCAs have to be screened for their antagonist efficacy against Plasmopara viticola. Evaluation of their effect on the pathogen can be achieved by detecting the amount of P. viticola DNA in leaves treated with potential antagonists and infected with the pathogen. In this study, a rapid high-throughput method was developed for relative quantification of P. viticola DNA directly from Vitis vinifera leaves by means of multiplex real-time quantitative polymerase chain reaction (PCR) with TaqMan chemistry. This method allows simultaneous amplification, but independent detection, of pathogen and host DNA by using species-specific primers and TaqMan probes that are labeled with different fluorescent dyes. Including detection of V. vinifera DNA in the tests is fundamental because it provides an endogenous reference and allows normalization for variations caused by sample-to-sample differences in DNA extraction, PCR efficiencies, and pipetting volumes. The developed method allows highly sensitive and specific detection of P. viticola DNA (minimal detectable quantity of 0.1 pg). Moreover, high precision and reproducibility of TaqMan assays were observed over a linear range of four orders of magnitude, confirming the reliability of the developed PCR assay. Potential applications range from screening for BCA efficiency to evaluation of fungicide efficacy, or assessment of host resistance.

Additional keywords: biocontrol agents , downy mildew , TaqMan .